Indiana University-Purdue University Indianapolis (IUPUI). / Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical modulator of synaptic function, plasticity, and learning and memory. In neurons and astrocytes, CaMKII regulates cellular excitability, cytoskeletal structure, and cell metabolism. A rapid increase in CaMKII activity is observed within the first few minutes of ischemic stroke in vivo; this calcium-dependent process is also observed following glutamate stimulation in vitro. Activation of CaMKII during pathological conditions is immediately followed by inactivation and aggregation of the kinase. The extent of CaMKII inactivation is directly correlated with the extent of neuronal damage. The studies presented here show that these fluctuations in CaMKII activity are not correlated with neuronal death; rather, they play a causal role in neuronal death. Pharmacological inhibition of CaMKII in the time immediately surrounding glutamate insult protects cultured cortical neurons from excitotoxicity. Interestingly, pharmacological inhibition of CaMKII during excitotoxic insult also prevents the aggregation and prolonged inactivation of the kinase, suggesting that CaMKII activity during excitotoxic glutamate signaling is detrimental to neuronal viability because it leads to a prolonged loss of CaMKII activity, culminating in neuronal death. In support of this, CaMKII inhibition in the absence of excitotoxic insult induces cortical neuron apoptosis by dysregulating intracellular calcium homeostasis and increasing excitatory glutamate signaling. Blockade of the NMDA-receptors and enzymatic degradation of the extracellular glutamate signal affords neuroprotection from CaMKII inhibition-induced toxicity. Co-cultures of neurons and glutamate-buffering astrocytes also exhibit this slow-induced excitotoxicity, as CaMKII inhibitors reduce glutamate uptake within the astrocytes. CaMKII inhibition also dysregulates calcium homeostasis in astrocytes and leads to increased ATP release, which was neurotoxic when applied to naïve cortical neurons. Together, these findings indicate that during aberrant calcium signaling, the activation of CaMKII is toxic because it supports aggregation and prolonged inactivation of the kinase. Without CaMKII activity, neurons and astrocytes release stores of transmitters that further exacerbate neuronal toxicity.

Previous nutritional physiology research using L-histidine and zinc in American lobster intestine (Homarus americanus) has suggested that these solutes can be co-transported as complexes (Histidine-Zinc-Histidine) across the intestine using a peptide transporter. Furthermore, transport of L-leucine was shown to be inhibited by high calcium concentrations. Dipeptide and bis-complex transport and the role of calcium were investigated in the perfused intestines of lobster and Atlantic white shrimp (Litopenaeus setiferus). Following trans-intestinal transport, serosal medium was analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly-Sar) from mucosa to serosa was stimulated two-fold with luminal pH 8.5, compared to the pH 5.5 control. Mucosa to serosa and serosa to mucosa fluxes of Gly-Sar were measured; the dipeptide was transported intact in both directions, but the net flux was from mucosa to serosa. The use of 0.5mM calcium chloride stimulated Gly-Sar transport two-fold, compared to 25 mM. In shrimp, the addition of 50 µM zinc chloride increased the rate of L-histidine transport, while Gly-Sar inhibited histidine transport in the presence of zinc. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results suggest that shrimp transport bis-complexes in a manner similar to lobster. High calcium concentration had an inhibitory effect on both amino acid and dipeptide transport. Proposed mechanisms accounting for the effects of metals and calcium on trans-intestinal transports of both amino acids and dipeptides by lobster and shrimp digestive tracts are discussed.

Indiana University-Purdue University Indianapolis (IUPUI) / For years, the leading killer of Americans has been coronary artery disease (CAD), which has a strong correlation to the U.S. obesity epidemic. Obesity, along with the presence of other risk factors including hyperglycemia, hypercholesterolemia, dyslipidemia, and high blood pressure, comprise of the diagnosis of metabolic syndrome (MetS). The presentation of multiple MetS risk factors increases a patients risk for adverse cardiovascular events. CAD is a complex progressive disease. We utilized the superb model of CAD and MetS, the Ossabaw miniature swine, to investigate underlying mechanisms of CAD progression. We studied the influence of coronary epicardial adipose tissue (cEAT) and coronary smooth muscle cell (CSM) intracellular Ca2+ regulation on CAD progression. By surgical excision of cEAT from MetS Ossabaw, we observed an attenuation of CAD progression. This finding provides evidence for a link between local cEAT and CAD progression. Intracellular Ca2+ is a tightly regulated messenger in CSM that initiates contraction, translation, proliferation and migration. When regulation is lost, CSM dedifferentiate from their mature, contractile phenotype found in the healthy vascular wall to a synthetic, proliferative phenotype. Synthetic CSM are found in intimal plaque of CAD patients. We investigated the changes in intracellular Ca2+ signaling in enzymatically isolated CSM from Ossabaw swine with varying stages of CAD using the fluorescent Ca2+ indicator, fura-2. This time course study revealed heightened Ca2+ signaling in early CAD followed by a significant drop off in late stage calcified plaque. Coronary artery calcification (CAC) is a result of dedifferentiation into an osteogenic CSM that secretes hydroxyapatite in the extracellular matrix. CAC is clinically detected by computed tomography (CT). Microcalcifications have been linked to plaque instability/rupture and cannot be detected by CT. We used 18F-NaF positron emission tomography (PET) to detect CAC in Ossabaw swine with early stage CAD shown by mild neointimal thickening. This study validated 18F-NaF PET as a diagnostic tool for early, molecular CAC at a stage prior to lesions detectable by CT. This is the first report showing non-invasive PET resolution of CAC and CSMC Ca2+ dysfunction at an early stage previously only characterized by invasive cellular Ca2+ imaging.